Personal of Sirt3 making use of quantitative PCR and western blot, respectively. d Acetylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 working with western blot analysis. e Nitrosylation of SOD2, precipitated from HAEC following transient knockdown of Sirt3 utilizing western blot analysis. f Expression analyses of f catalase, g SOD1, h SOD3, using quantitative PCR; beta actin served as loading manage in western blots, representative blots are shown. At least three independent experiments in biological triplicates had been performed, scr scrambled controlPage 7 of(A)SOD2-specific activity (normalized to mg protein)1.p0.(B)SOD2 activity (unadjusted)p=0.enzymatic activity per mg protein [AU]enzymatic activity [AU]2.0 1.5 1.0 0.five 0.r sc1.0.0.r rt3 sc Si sirelative protein expression(C)relative mRNA expression6 5 4 3 two 1rSODp0.four 3 two 1r scp=0.SOD2 actinr rtrt3 sc si r Si rtrtscscSiscsiSisisiSirsiSirtsiSirt(D)relative protein acetylation(E)relative nitrosylation [AU]SOD2 nitrosylation1.p=1.SOD2 acetylation4.0 3.0 two.0 1.0 0.r rt3 sc Si sianti-acK IP: SOD2 p=0.1.0.r sc si Si rtr scsiSirt(F)relative mRNA expression(G)relative mRNA expression(H)relative mRNA expressionCatalase1.p=0.SOD1.p=0.SOD2.0 1.5 1.0 0.5 0.r sc si Si rtp=0.1.1.0.0.0.r rt3 sc Si si0.r sc si Si rtTranscriptional induction of SOD2 upon transient knockdown of Sirt3 is C/EBP-b-dependent To shed light on the unexpected transcriptional upregulation of endothelial SOD2 during transient knockdown of Sirt3, we assessed the expression levels of recognized, acetylation-dependent transcription elements of SOD2. Expressionlevels of Sp1 and STAT3 have been unaffected by transient knockdown of Sirt3 (Fig. 4a, b), whereas expression of CCAAT/enhancer binding protein beta (C/EBP-b) was increased (Fig. 4c). This differential expression in response to decreased Sirt3 levels prompted us to address the relevance of C/EBP-b within the transcriptional induction of SOD2 duringPage eight ofBasic Res Cardiol (2016) 111:(A)relative mRNA expression1.Spp=0.(B)relative mRNA expression2.0 1.5 1.0 0.five 0.STATp=0.(C)relative mRNA expression2.5 two.0 1.5 1.0 0.5 0.C/EBP-p0.1.0.0.rrtscscrtscSiSisisi(D)relative mRNA expression2.0 1.five 1.0 0.five 0.C/EBP-C/EBP-relative protein expression**** **** ****n.2-Chloro-5-hydroxyisonicotinic acid site s.Imidazo[1,2-a]pyridine-8-carbaldehyde web (E)two.five two.0 1.5 1.0 0.five 0.*** **** *C/EBP-actinrr-crrcrsiSi-scsc/ssc/s/sBP/sBPr/r/rtr/rtrt-/E/EscscBPiCsciCSiSi/sSi/sC/ErtsirtSiSisi(F)relative mRNA expression8.0 6.0 four.SODn.s.si(G)relative protein expression2.0 1.5 1.0 0.five 0.SOD*n.s.** n.s. **2.0 0.SOD2 actinrcrsiSirt/s /s Si rt3 /s iC /E BP -sisisc si r / s si Si Si rt3 cr rt3 / /s iC scr /E BP -rcr-crsc/s/sBPr/r/scrtscBPsc/ESiiCC/EsiSirt/ssisiSiFig. four Transcriptional induction of SOD2 upon transient knockdown of Sirt3 is C/EBP-b-dependent.PMID:24059181 a Expression analyses from the transcription components a Sp1, b STAT3, c, d C/EBP-b utilizing quantitative PCR in HAEC following siRNA-mediated knockdown of Sirt3, and d single or simultaneous transient knockdown of C/EBP-b and Sirt3, respectively. e Western blot evaluation of C/EBP-b in HAEC following single and simultaneous transient knockdown of C/EBP-b and Sirt3, respectively. f, g Expression analyses of SOD2 in HAEC followingsisingle or simultaneous transient knockdown of C/EBP-b and Sirt3, respectively, working with f quantitative PCR and g western blot analysis. At least 3 independent experiments in biological triplicates were performed, scr scrambled handle, d p values indicate overall significance by Kruskal allis (d ) or one-way ANOVA tests (g),.